Burkholderia pseudomallei BipD modulates host mitophagy to evade killing.

Mitophagy is critical for mitochondrial quality control and function to clear damaged mitochondria. Here, we found that Burkholderia pseudomallei maneuvered host mitophagy for its intracellular survival through the type III secretion system needle tip protein BipD. We identified BipD, interacting with BTB-containing proteins KLHL9 and KLHL13 by binding to the Back and Kelch domains, recruited NEDD8 family RING E3 ligase CUL3 in response to B. pseudomallei infection. Although evidently not involved in regulation of infectious diseases, KLHL9/KLHL13/CUL3 E3 ligase complex was essential for BipD-dependent ubiquitination of mitochondria in mouse macrophages. Mechanistically, we discovered the inner mitochondrial membrane IMMT via host ubiquitome profiling as a substrate of KLHL9/KLHL13/CUL3 complex. Notably, K63-linked ubiquitination of IMMT K211 was required for initiating host mitophagy, thereby reducing mitochondrial ROS production. Here, we show a unique mechanism used by bacterial pathogens that hijacks host mitophagy for their survival.

TLR9 Negatively Regulates Intracellular Bacterial Killing by Pyroptosis in Burkholderia pseudomallei-Infected Mouse Macrophage Cell Line (Raw264.7).

Melioidosis is a serious infectious disease caused by Burkholderia pseudomallei. This bacterium is able to survive and multiply inside the immune cells such as macrophages. It is well established that Toll-like receptors (TLRs), particularly surface TLRs such as TLR2, TLR4, and TLR5, play an essential role in defending against this bacterial infection. However, the involvement of endosomal TLRs in the infection has not been elucidated. In this study, we demonstrated that the number of intracellular bacteria is reduced in TLR9-depleted RAW264.7 cells infected with B. pseudomallei, suggesting that TLR9 is involved in intracellular bacterial killing in macrophages. As several reports have previously demonstrated that pyroptosis is essential for restricting intracellular bacterial killing, particularly in B. pseudomallei infection, we also observed an increased release of cytosolic enzyme lactate dehydrogenase (LDH) in TLR9-depleted cells infected with B. pseudomallei, suggesting TLR9 involvement in pyroptosis in this context. Consistently, the increases in caspase-11 and gasdermind D (GSDMD) activations, which are responsible for the LDH release, were also detected. Moreover, we demonstrated that the increases in pyroptosis and bacterial killing in B. pseudomallei-infected TLR9-depleted cells were due to the augmentation of the IFN-ß, one of the key cytokines known to regulate caspase-11. Altogether, this finding showed that TLR9 suppresses macrophage killing of B. pseudomallei by regulating pyroptosis. This information provides a novel mechanism of TLR9 in the regulation of intracellular bacterial killing by macrophages, which could potentially be leveraged for therapeutic intervention. IMPORTANCE Surface TLRs have been well established to play an essential role in Burkholderia pseudomallei infection. However, the role of endosomal TLRs has not been elucidated. In the present study, we demonstrated that TLR9 plays a crucial role by negatively regulating cytokine production, particularly IFN-ß, a vital cytokine to control pyroptosis via caspase-11 activation. By depletion of TLR9, the percentage of pyroptosis was significantly increased, leading to suppression of intracellular survival in B. pseudomallei-infected macrophages. These findings provide a new role of TLR9 in macrophages.

Dual RNA-seq reveals a type 6 secretion system-dependent blockage of TNF-α signaling and BicA as a Burkholderia pseudomallei virulence factor important during gastrointestinal infection.

Melioidosis is a disease caused by the Gram-negative bacillus Burkholderia pseudomallei (Bpm), commonly found in soil and water of endemic areas. Naturally acquired human melioidosis infections can result from either exposure through percutaneous inoculation, inhalation, or ingestion of soil-contaminated food or water. Our prior studies recognized Bpm as an effective enteric pathogen, capable of establishing acute or chronic gastrointestinal infections following oral inoculation. However, the specific mechanisms and virulence factors involved in the pathogenesis of Bpm during intestinal infection are unknown. In our current study, we standardized an in vitro intestinal infection model using primary intestinal epithelial cells (IECs) and demonstrated that Bpm requires a functional T6SS for full virulence. Further, we performed dual RNA-seq analysis on Bpm-infected IECs to evaluate differentially expressed host and bacterial genes in the presence or absence of a T6SS. Our results showed a dysregulation in the TNF-α signaling via NF-κB pathway in the absence of the T6SS, with some of the genes involved in inflammatory processes and cell death also affected. Analysis of the bacterial transcriptome identified virulence factors and regulatory proteins playing a role during infection, with association to the T6SS. By using a Bpm transposon mutant library and isogenic mutants, we showed that deletion of the bicA gene, encoding a putative T3SS/T6SS regulator, ablated intracellular survival and plaque formation by Bpm and impacted survival and virulence when using murine models of acute and chronic gastrointestinal infection. Overall, these results highlight the importance of the type 6 secretion system in the gastrointestinal pathogenesis of Bpm.

Bioinformatics Analysis for Identifying Pertinent Pathways and Genes in Sepsis.

PURPOSE: Sepsis becomes the main death reason in hospitals with rising incidence, causing a growing economic and medical burden. However, the genes related to the pathogenesis and prognosis of sepsis are still unclear, which is a problem that needs to be solved urgently. MATERIALS AND METHODS: Gene expression profiles of GSE69528 were obtained from the National Center for Biotechnology Information. Limma software package got employed to search for differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) were used for enrichment analysis. Protein-protein interaction (PPI) network was built by the Search Tool for the Retrieval of Interacting Genes (STRING) database. RESULTS: We screened 101 DEGs, containing 81 upregulated DEGs and 20 downregulated DEGs. GO analysis demonstrated that the upregulated DEGs were chiefly concentrated in negative regulation of response to interferon-gamma and regulation of granulocyte differentiation. KEGG analysis revealed that the pathways of upregulated DEGs were concentrated in prion diseases, complement and coagulation cascades, and Staphylococcus aureus infection. The PPI network constructed by upregulated DEGs contained 67 nodes (proteins) and 110 edges (interactions). Analysis of bioinformatics results showed that CEACAM8, MPO, and RETN were hub genes of sepsis. CONCLUSION: Our analysis reveals a series of signal pathways and key genes related to the mechanism of sepsis, which are promising biotargets and biomarkers of sepsis.

Skin models for cutaneous melioidosis reveal Burkholderia infection dynamics at wound's edge with inflammasome activation, keratinocyte extrusion and epidermal detachment.

ABSTRACTMelioidosis is a serious infectious disease endemic in Southeast Asia, Northern Australia and has been increasingly reported in other tropical and subtropical regions in the world. Percutaneous inoculation through cuts and wounds on the skin is one of the major modes of natural transmission. Despite cuts in skin being a major route of entry, very little is known about how the causative bacterium Burkholderia pseudomallei initiates an infection at the skin and the disease manifestation at the skin known as cutaneous melioidosis. One key issue is the lack of suitable and relevant infection models. Employing an in vitro 2D keratinocyte cell culture, a 3D skin equivalent fibroblast-keratinocyte co-culture and ex vivo organ culture from human skin, we developed infection models utilizing surrogate model organism Burkholderia thailandensis to investigate Burkholderia-skin interactions. Collectively, these models show that the bacterial infection was largely limited at the wound's edge. Infection impedes wound closure, triggers inflammasome activation and cellular extrusion in the keratinocytes as a potential way to control bacterial infectious load at the skin. However, extensive infection over time could result in the epidermal layer being sloughed off, potentially contributing to formation of skin lesions.

In silico prediction of host-pathogen protein interactions in melioidosis pathogen Burkholderia pseudomallei and human reveals novel virulence factors and their targets.

The aerobic, Gram-negative motile bacillus, Burkholderia pseudomallei is a facultative intracellular bacterium causing melioidosis, a critical disease of public health importance, which is widely endemic in the tropics and subtropical regions of the world. Melioidosis is associated with high case fatality rates in animals and humans; even with treatment, its mortality is 20-50%. It also infects plants and is designated as a biothreat agent. B. pseudomallei is pathogenic due to its ability to invade, resist factors in serum and survive intracellularly. Despite its importance, to date only a few effector proteins have been functionally characterized, and there is not much information regarding the host-pathogen protein-protein interactions (PPI) of this system, which are important to studying infection mechanisms and thereby develop prevention measures. We explored two computational approaches, the homology-based interolog and the domain-based method, to predict genome-scale host-pathogen interactions (HPIs) between two different strains of B. pseudomallei (prototypical, and highly virulent) and human. In total, 76 335 common HPIs (between the two strains) were predicted involving 8264 human and 1753 B. pseudomallei proteins. Among the unique PPIs, 14 131 non-redundant HPIs were found to be unique between the prototypical strain and human, compared to 3043 non-redundant HPIs between the highly virulent strain and human. The protein hubs analysis showed that most B. pseudomallei proteins formed a hub with human dnaK complex proteins associated with tuberculosis, a disease similar in symptoms to melioidosis. In addition, drug-binding and carbohydrate-binding mechanisms were found overrepresented within the host-pathogen network, and metabolic pathways were frequently activated according to the pathway enrichment. Subcellular localization analysis showed that most of the pathogen proteins are targeting human proteins inside cytoplasm and nucleus. We also discovered the host targets of the drug-related pathogen proteins and proteins that form T3SS and T6SS in B. pseudomallei. Additionally, a comparison between the unique PPI patterns present in the prototypical and highly virulent strains was performed. The current study is the first report on developing a genome-scale host-pathogen protein interaction networks between the human and B. pseudomallei, a critical biothreat agent. We have identified novel virulence factors and their interacting partners in the human proteome. These PPIs can be further validated by high-throughput experiments and may give new insights on how B. pseudomallei interacts with its host, which will help medical researchers in developing better prevention measures.

Predicting toxins found in toxin-antitoxin systems with a role in host-induced Burkholderia pseudomallei persistence.

Burkholderia pseudomallei (Bpm) is a bacterial pathogen that causes Melioidosis, a disease with up to 40% mortality and an infection relapse of 15-23% despite antibiotic treatment. Ineffective clearance of Bpm by antibiotics is believed to be due to persistence, a hibernation-like survival mechanism modulated, in part, by toxin-antitoxin systems (TAS). Several organisms possess a repertoire of TASs but defining environmental cues eliciting their activity is hindered by laborious in vitro experiments, especially when there are many toxins with redundant function. Here, we identified which of 103 proteins in Bpm that share features found in toxins of the TAS and repurposed transcriptional data to identify which ones play a role in surviving intracellular host defenses. Putative toxins with the strongest transcriptional response were found to have low conservation between Bpm strains, while toxins that were constitutively expressed were highly conserved. Further examination of highly conserved toxins BPSS0899, BPSS1321, and BPSL1494 showed that they were functional, and their mutation led to reduce survival within macrophages and reduced in vivo persistence-associated pathology (abscesses) during treatment, but did not affect macrophages persistence. These findings highlight the utility of a data-driven approach to select putative toxins and suggests a selective role for some TAS in host survival.

An increase in p62/NBR1 levels in melioidosis patients of Sri Lanka exhibit a characteristic of potential host biomarker.

Introduction. Melioidosis, caused by Burkholderia pseudomallei, in endemic areas, poses a challenge for treating the diseased populations without accurate diagnosis, and the disease-specific biomarkers linked with the infection have yet to be reported. Due to the invasive nature of the causative agent, Burkholderia pseudomallei, host innate effector mechanisms, including autophagy are known to be activated, resulting in differential expression of cellular proteins and immune markers. Identification of a disease-specific biomarker associated with B. pseudomallei infection will be helpful to facilitate rapid confirmation of melioidosis, which would enable early treatment and therapeutic success.Aim. We aimed to assess the levels of a host autophagy component, p62/NBR1, which function as a cargo-receptor in the process of autophagy activation leading to the degradation of ubiquitin-coated intracellular bacteria in which p62/NBR1 itself is degraded in the clearance of the pathogen. We further probed the extent of intracellular p62/NBR1 degradation and assessed its potential as a melioidosis biomarker.Methodology. We analysed peripheral blood mononuclear cell (PBMC) lysates using an ELISA-based assay for detecting cytosolic autophagy-related proteins p62/NBR1. We measured p62/NBR1 levels in diseased (confirmed B. pseudomallei infection) and non -diseased populations and utilized receiver operating characteristic (ROC) curve and max Youden index analysis for evaluating potential disease biomarker characteristics.Results. Our results revealed a three to fivefold increase in p62/NBR1 levels confirmed melioidosis cases compared to uninfected healthy donors. Comparable to p62/NBR1, levels of cytosolic LC3-I levels also increased, whereas the levels of degraded membrane bound form LC3-II was low, suggesting autophagy deficiency. Proinflammatory serum cytokine response, particularly IL-6, was consistently higher alongside B. pseudomallei infection in comparison to healthy controls.Conclusions. ROC curve and max Youden index analysis suggest that increased p62/NBR1 levels in diseased populations display characteristics of a potential disease biomarker in melioidosis and illustrates that an elevated p62/NBR1 level, in conjunction with B. pseudomallei infection associated with autophagy deficiency.

Lactoferrin is a dynamic protein in human melioidosis and is a TLR4-dependent driver of TNF-α release in Burkholderia thailandensis infection in vitro.

Melioidosis is an often-severe tropical infection caused by Burkholderia pseudomallei (Bp) with high associated morbidity and mortality. Burkholderia thailandensis (Bt) is a closely related surrogate that does not require BSL-3 conditions for study. Lactoferrin is an iron-binding glycoprotein that can modulate the innate inflammatory response. Here we investigated the impact of lactoferrin on the host immune response in melioidosis. Lactoferrin concentrations were measured in plasma from patients with melioidosis and following ex vivo stimulation of blood from healthy individuals. Bt growth was quantified in liquid media in the presence of purified and recombinant human lactoferrin. Differentiated THP-1 cells and human blood monocytes were infected with Bt in the presence of purified and recombinant human lactoferrin, and bacterial intracellular replication and cytokine responses (tumor necrosis factor-α (TNF-α), interleukin-1ß and interferon-γ) were measured. In a cohort of 49 melioidosis patients, non-survivors to 28 days had significantly higher plasma lactoferrin concentrations compared to survivors (median (interquartile range (IQR)): 326 ng/ml (230-748) vs 144 ng/ml (99-277), p<0.001). In blood stimulated with heat-killed Bp, plasma lactoferrin concentration significantly increased compared to unstimulated blood (median (IQR): 424 ng/ml (349-479) vs 130 ng/ml (91-214), respectively; p<0.001). Neither purified nor recombinant human lactoferrin impaired growth of Bt in media. Lactoferrin significantly increased TNF-α production by differentiated THP-1 cells and blood monocytes after Bt infection. This phenotype was largely abrogated when Toll-like receptor 4 (TLR4) was blocked with a monoclonal antibody. In sum, lactoferrin is produced by blood cells after exposure to Bp and lactoferrin concentrations are higher in 28-day survivors in melioidosis. Lactoferrin induces proinflammatory cytokine production after Bt infection that may be TLR4 dependent.

Transcriptional profiling unveils type I and II interferon networks in blood and tissues across diseases.

Understanding how immune challenges elicit different responses is critical for diagnosing and deciphering immune regulation. Using a modular strategy to interpret the complex transcriptional host response in mouse models of infection and inflammation, we show a breadth of immune responses in the lung. Lung immune signatures are dominated by either IFN-γ and IFN-inducible, IL-17-induced neutrophil- or allergy-associated gene expression. Type I IFN and IFN-γ-inducible, but not IL-17- or allergy-associated signatures, are preserved in the blood. While IL-17-associated genes identified in lung are detected in blood, the allergy signature is only detectable in blood CD4+ effector cells. Type I IFN-inducible genes are abrogated in the absence of IFN-γ signaling and decrease in the absence of IFNAR signaling, both independently contributing to the regulation of granulocyte responses and pathology during Toxoplasma gondii infection. Our framework provides an ideal tool for comparative analyses of transcriptional signatures contributing to protection or pathogenesis in disease.

An increase in intracellular p62/NBR1 and persistence of Burkholderia mallei and B. pseudomallei in infected mice linked to autophagy deficiency.

INTRODUCTION: Burkholderia mallei (B. mallei) and Burkholderia pseudomallei (B. pseudomallei), causative agents of glanders and melioidosis, respectively, are invasive intracellular pathogens that actively multiply in phagocytic and non-phagocytic cells. Activation of cell-autonomous autophagy mechanism eliminate intracellular pathogens in which p62 a cytosolic cargo protein is selectively degraded, and an accumulation of this marker occurs if autophagy is deficient. Recurrent, relapsed and reinfection of B. pseudomallei in melioidosis patients in endemic area indicative of lack of complete of clearance and persistence of the pathogen. Reasoning that abundance in the levels of p62 may provide an indication of the intracellular infection, we sought to examine whether increase in intracellular p62 and bacterial burden with Burkholderia infection are linked to autophagy deficiency. METHODS: In this study, we investigated cell culture and mouse models of disease to identify an association between autophagy biomarkers (p62/NBR1) accumulation and intracellular persistence of B. mallei and B. pseudomallei. RESULTS: We demonstrate, that elevated levels of intracellular p62/NBR1 correlated with bacterial persistence, while pre-treatment with a pharmacological inducer of autophagy, rapamycin, reduced both intracellular p62, and bacterial survival. Our results showed an elevated p62 levels (2-5 fold) in spleen and liver cells of Burkholderia-infected BALB/c mice, as well as in spleen cells of Burkholderia-infected C57BL/6 mice, suggesting that an increase in p62/NBR1 was due to an autophagy deficiency. Similar to p62, cytosolic LC3-I levels were also elevated, while the characteristic conversion to the autophagosome-associated membrane bound form LC3-II was low in spleens of the infected mice further supporting the conclusion that autophagy was deficient. CONCLUSION: Taken together, our results suggest that an increase in intracellular p62/NBR1 may be a potential host cell biomarker of B. mallei or B. pseudomallei infections, and identifying autophagy manipulation may potentially aid to therapeutic approach for complete clearance of the pathogen.

Upregulation of Immune Process-Associated Genes in RAW264.7 Macrophage Cells in Response to Burkholderia pseudomallei Infection.

Melioidosis is a severe and fatal tropical zoonosis, which is triggered by Burkholderia pseudomallei. To better understand the host's response to infection of B. pseudomallei, an RNA-Seq technology was used to confirm differentially expressed genes (DEGs) in RAW264.7 cells infected with B. pseudomallei. In total, 4668 DEGs were identified across three time points (4, 8, and 11 hours after infection). Short Time-Series Expression Miner (STEM) analysis revealed the temporal gene expression profiles and identified seven significant patterns in a total of 26 profiles. Kyoto Encyclopedia of Genes and Genomes (KEGG) was utilized to confirm significantly enriched immune process-associated pathways, and 10 DEGs, including Ccl9, Ifnb1, Tnfα, Ptgs2, Tnfaip3, Zbp1, Ccl5, Ifi202b, Nfkbia, and Nfkbie, were mapped to eight immune process-associated pathways. Subsequent quantitative real-time PCR assays confirmed that the 10 DEGs were all upregulated during infection. Overall, the results showed that B. pseudomallei infection can initiate a time-series upregulation of immune process-associated DEGs in RAW264.7 macrophage cells. The discovery of this article helps us better understand the biological function of the immune process-associated genes during B. pseudomallei infection and may aid in the development of prophylaxis and treatment protocols for melioidosis.

Lipid A Remodeling Is a Pathoadaptive Mechanism That Impacts Lipopolysaccharide Recognition and Intracellular Survival of Burkholderia pseudomallei.

Burkholderia pseudomallei causes the severe disease melioidosis. The bacterium subverts the host immune system and replicates inside cells, and host mortality results primarily from sepsis-related complications. Lipopolysaccharide (LPS) is a major virulence factor and mediator of sepsis that many pathogens capable of intracellular growth modify to reduce their immunological "footprint." The binding strength of B. pseudomallei LPS for human LPS binding protein (hLBP) was measured using surface plasmon resonance. The structures of lipid A isolated from B. pseudomallei under different temperatures were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and the gene expression of two lipid A remodeling genes, lpxO and pagL, was investigated. The LPS was characterized for its ability to trigger tumor necrosis factor alpha (TNF-α) release and to activate caspase-11-triggered pyroptosis by introduction of LPS into the cytosol. Lipid A from long-term chronic-infection isolates was isolated and characterized by MALDI-TOF MS and also by the ability to trigger caspase-11-mediated cell death. Lipid A from B. pseudomallei 1026b lpxO and pagL mutants were characterized by positive- and negative-mode MALDI-TOF MS to ultimately identify their role in lipid A structural modifications. Replication of lpxO and pagL mutants and their complements within macrophages showed that lipid A remodeling can effect growth in host cells and activation of caspase-11-mediated cytotoxicity.

Adhesion and invasion attributes of Burkholderia pseudomallei are dependent on airway surface liquid and glucose concentrations in lung epithelial cells.

Physiological constituents in airway surface liquids (ASL) appear to impact the adherence and invasion potentials of Burkholderia pseudomallei contributing to recrudescent melioidosis. Here, we investigated the factors present in ASL that is likely to influence bacterial adhesion and invasion leading to improved understanding of bacterial pathogenesis. Six B. pseudomallei clinical isolates from different origins were used to investigate the ability of the bacteria to adhere and invade A549 human lung epithelial cells using a system that mimics the physiological ASL with different pH, NaCl, KCl, CaCl2 and glucose concentrations. These parameters resulted in markedly differential adherence and invasion abilities of B. pseudomallei to the lung epithelial cells. The concentration of 20 mM glucose dramatically increased adherence and invasion by increasing the rate of pili formation in depiliated bacteria. Glucose significantly increased adherence and invasion of B. pseudomallei to A549 cells, and presence of NaCl, KCl and CaCl2 markedly ablated the effect despite the presence of glucose. Our data established a link between glucose, enhanced adhesion and invasion potentials of B. pseudomallei, hinting increased susceptibility of individuals with diabetes mellitus to clinical melioidosis.

Type III Secretion in the Melioidosis Pathogen Burkholderia pseudomallei.

Burkholderia pseudomallei is a Gram-negative intracellular pathogen and the causative agent of melioidosis, a severe disease of both humans and animals. Melioidosis is an emerging disease which is predicted to be vastly under-reported. Type III Secretion Systems (T3SSs) are critical virulence factors in Gram negative pathogens of plants and animals. The genome of B. pseudomallei encodes three T3SSs. T3SS-1 and -2, of which little is known, are homologous to Hrp2 secretion systems of the plant pathogens Ralstonia and Xanthomonas. T3SS-3 is better characterized and is homologous to the Inv/Mxi-Spa secretion systems of Salmonella spp. and Shigella flexneri, respectively. Upon entry into the host cell, B. pseudomallei requires T3SS-3 for efficient escape from the endosome. T3SS-3 is also required for full virulence in both hamster and murine models of infection. The regulatory cascade which controls T3SS-3 expression and the secretome of T3SS-3 have been described, as well as the effect of mutations of some of the structural proteins. Yet only a few effector proteins have been functionally characterized to date and very little work has been carried out to understand the hierarchy of assembly, secretion and temporal regulation of T3SS-3. This review aims to frame current knowledge of B. pseudomallei T3SSs in the context of other well characterized model T3SSs, particularly those of Salmonella and Shigella.

Epidemiological cut-off value of clinical isolates of Burkholderia pseudomallei from Northern Queensland to meropenem, ceftazidime, trimethoprim/sulfamethoxazole and doxycycline by the microbroth dilution method.

OBJECTIVES: Melioidosis is caused by the bacterium Burkholderia pseudomallei. The most common antibiotics used to treat melioidosis in Australia are meropenem, ceftazidime, trimethoprim/sulfamethoxazole (SXT) and doxycycline. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) do not provide standards for assessing the susceptibility of B. pseudomallei for these agents. The International Standards Organisation (ISO) microbroth dilution method is accepted both by the CLSI and EUCAST as the gold standard of antimicrobial susceptibility testing. Many previous studies of the susceptibility of B. pseudomallei used Etest or disk diffusion and presented the results as aggregate data. Etest and disk diffusion methods have not been standardised for B. pseudomallei and aggregate data cannot be used to determine an epidemiological cut-off value (ECOFF). An ECOFF is vital for the setting of clinical breakpoints. METHODS: In this study, minimum inhibitory concentrations (MICs) of meropenem, ceftazidime, SXT and doxycycline were assessed by microbroth dilution for a library of 234 well characterised clinical isolates of B. pseudomallei from Northern Queensland, Australia. RESULTS: The resultant histograms and aggregate data represent the first MIC profile of a large library of B. pseudomallei that has been successfully produced using microbroth dilution. CONCLUSIONS: The MIC profiles can be used to contribute towards a determination of an ECOFF for this species for these agents, which will aid in the setting and refining of clinical breakpoints for the most important antimicrobials used to treat melioidosis.

Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif).

Cycle inhibiting factors (Cifs) are virulence proteins secreted by the type III secretion system of some Gram-negative pathogenic bacteria including Burkholderia pseudomallei. Cif is known to function to deamidate Nedd8, leading to inhibition of Cullin E3 ubiquitin ligases (CRL) and consequently induction of cell cycle arrest. Here we show that Cif can function as a potent activator of MAPK/ERK signaling without significant activation of other signaling pathways downstream of receptor tyrosine kinases. Importantly, we found that the ability of Cif to activate ERK is dependent on its deamidase activity, but independent of Cullin E3 ligase inhibition. This suggests that apart from Nedd8, other cellular targets of Cif-dependent deamidation exist. We provide evidence that the mechanism involved in Cif-mediated ERK activation is dependent on recruitment of the Grb2-SOS1 complex to the plasma membrane. Further investigation revealed that Cif appears to modify the phosphorylation status of SOS1 in a region containing the CDC25-H and proline-rich domains. It is known that prolonged Cullin E3 ligase inhibition leads to cellular apoptosis. Therefore, we hypothesize that ERK activation is an important mechanism to counter the pro-apoptotic effects of Cif. Indeed, we show that Cif dependent ERK activation promotes phosphorylation of the proapoptotic protein Bim, thereby potentially conferring a pro-survival signal. In summary, we identified a novel deamidation-dependent mechanism of action of the B. pseudomallei virulence factor Cif/CHBP to activate MAPK/ERK signaling. Our study demonstrates that bacterial proteins such as Cif can serve as useful molecular tools to uncover novel aspects of mammalian signaling pathways.

Survival and Intra-Nuclear Trafficking of Burkholderia pseudomallei: Strategies of Evasion from Immune Surveillance?

BACKGROUND: During infection, successful bacterial clearance is achieved via the host immune system acting in conjunction with appropriate antibiotic therapy. However, it still remains a tip of the iceberg as to where persistent pathogens namely, Burkholderia pseudomallei (B. pseudomallei) reside/hide to escape from host immune sensors and antimicrobial pressure. METHODS: We used transmission electron microscopy (TEM) to investigate post-mortem tissue sections of patients with clinical melioidosis to identify the localisation of a recently identified gut microbiome, B. pseudomallei within host cells. The intranuclear presence of B. pseudomallei was confirmed using transmission electron microscopy (TEM) of experimentally infected guinea pig spleen tissues and Live Z-stack, and ImageJ analysis of fluorescence microscopy analysis of in vitro infection of A549 human lung epithelial cells. RESULTS: TEM investigations revealed intranuclear localization of B. pseudomallei in cells of infected human lung and guinea pig spleen tissues. We also found that B. pseudomallei induced actin polymerization following infection of A549 human lung epithelial cells. Infected A549 lung epithelial cells using 3D-Laser scanning confocal microscopy (LSCM) and immunofluorescence microscopy confirmed the intranuclear localization of B. pseudomallei. CONCLUSION: B. pseudomallei was found within the nuclear compartment of host cells. The nucleus may play a role as an occult or transient niche for persistence of intracellular pathogens, potentially leading to recurrrent episodes or recrudescence of infection.

Sulfonamide inhibition profile of the γ-carbonic anhydrase identified in the genome of the pathogenic bacterium Burkholderia pseudomallei the etiological agent responsible of melioidosis.

A new γ-carbonic anhydrase (CA, EC 4.1.1.1) was cloned and characterized kinetically in the genome of the bacterial pathogen Burkholderia pseudomallei, the etiological agent of melioidosis, an endemic disease of tropical and sub-tropical regions of the world. The catalytic activity of this new enzyme, BpsCAγ, is significant with a kcat of 5.3×105s-1 and kcat/Km of 2.5×107M-1×s-1 for the physiologic CO2 hydration reaction. The inhibition constant value for this enzyme for 39 sulfonamide inhibitors was obtained. Acetazolamide, benzolamide and metanilamide were the most effective (KIs of 149-653nM) inhibitors of BpsCAγ activity, whereas other sulfonamides/sulfamates such as ethoxzolamide, topiramate, sulpiride, indisulam, sulthiame and saccharin were active in the micromolar range (KIs of 1.27-9.56µM). As Burkholderia pseudomallei is resistant to many classical antibiotics, identifying compounds that interfere with crucial enzymes in the B. pseudomallei life cycle may lead to antibiotics with novel mechanisms of action.

Formulation and Characterization of Nanocluster Ceftazidime for the Treatment of Acute Pulmonary Melioidosis.

Melioidosis is an infectious disease caused by Burkholderia pseudomallei. The disease is responsible for a high proportion of human pneumonia and fatal bacteremia in the endemic areas of the world and is highly resistant to most commonly available antibiotics. Studies have shown that prophylactic antibiotic treatment, when administered 24 h following bacterial challenge, can prevent infection in a murine model. Prophylactic treatment against this disease using a pulmonary antibiotic formulation has not previously been examined, but may reduce the number of treatments required, allow for the delivery of higher doses, eliminate the need for intravenous administration, and help to minimize systemic side effects. Ceftazidime was formulated as a dry powder aerosol suitable for pulmonary delivery using previously developed NanoCluster dry powder technology. Pharmacokinetics of aerosolized ceftazidime was analyzed in a mouse model. This study demonstrates that ceftazidime can be formulated using NanoCluster technology as a dry powder aerosol suitable for pulmonary delivery to humans. We have also demonstrated the retention of nebulized ceftazidime in mouse lungs for up to 6 h after exposure. The results indicate that this treatment may be useful as a prophylactic treatment against melioidosis. Future work will examine the efficacy of this treatment against B. pseudomallei aerosol challenge.